Methods to treat pompe disease using a recombinant adeno-associated virus

ABSTRACT

A method to prevent, inhibit or treat one or more symptoms associated with a disease of the central nervous system by intrathecally, intracerebroventricularly or endovascularly administering a rAAV encoding a gene product associated with the disease, e.g., a mammal in which the gene product is absent or present at a reduced level relative to a mammal without the disease.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.15/717,450, filed Sep. 27, 2017, which is a continuation of U.S. patentapplication Ser. No. 14/889,750, filed Nov. 6, 2015, now U.S. Pat. No.9,827,295, which is a U.S. National Stage filing under 35 U.S.C. 371from International Application No. PCT/US2014/038209, filed on May 15,2014 and published as WO 2014/186579 A1 on 20 Nov. 2014, which claimsthe benefit of the filing date of U.S. application Ser. No. 61/823,757,filed on May 15, 2013, the disclosures of which are incorporated byreference herein.

STATEMENT OF GOVERNMENT RIGHTS

This invention was made with government support under HD032652 andDK094538 awarded by National Institutes of Health. The Government hascertain rights in the invention.

BACKGROUND

The mucopolysaccharidoses (MPSs) are a group of 11 storage diseasescaused by disruptions in glycosaminoglycan (GAG) catabolism, leading totheir accumulation in lysosomes (Muenzer, 2004; Munoz-Rojas et al.,2008). Manifestations of varying severity include organomegaly, skeletaldysplasias, cardiac and pulmonary obstruction and neurologicaldeterioration. For MPS I, deficiency of iduronidase (IDUA), severityranges from mild (Scheie syndrome) to moderate (Hurler-Scheie) to severe(Hurler syndrome), with the latter resulting in neurologic deficiencyand death by age 15 (Muenzer, 2004; Munoz-Rojas et al., 2008). Therapiesfor MPSs have been for the most part palliative. However, there are someof the MPS diseases, including Hurler syndrome, for which allogeneichematopoietic stem cell transplantation (HSCT) has exhibited efficacy(Krivit, 2004; Orchard et al., 2007; Peters et al., 2003). Additionally,for more and more of the MPS diseases, enzyme replacement therapy (ERT)is becoming available (Brady, 2006). In general, HSCT and ERT result inthe clearing of storage materials and improved peripheral conditions,although some problems persist after treatment (skeletal, cardiac,corneal clouding). The primary challenge in these cellular and enzymetherapies is effectiveness in addressing neurological manifestations, asperipherally administered enzyme does not penetrate the blood-brainbarrier and HSCT has been found to be of benefit for some, but not all,MPS's.

MPS I has been one of the most extensively studied of the NAPS diseasesfor development of cellular and molecular therapies. The effectivenessof allogeneic HSCT is most likely the result of metaboliccross-correction, whereby the missing enzyme is released fromdonor-derived cells and subsequently taken up by host cells andtrafficked to lysosomes, where the enzyme contributes to lysosomalmetabolism (Fratantoni et al., 1968). Clearing of GAG storage materialsis subsequently observed in peripheral organs such as liver and spleen,there is relief from cardiopulmonary obstruction and improvement incorneal clouding (Orchard et al., 2007). Of particular importance is theeffect of allogeneic stem cell transplantation on the emergence ofneurologic manifestations in the MPS diseases. In this regard, there isevidence for several MPS diseases that individuals engrafted withallogeneic stem cells face an improved outcome in comparison withuntransplanted patients (Bjoraker et al., 2006; Krivit, 2004; Orchard etal., 2007; Peters et al., 2003). A central hypothesis explaining theneurologic benefit of allogeneic hematopoietic stem cell transplant isthe penetration of donor-derived hematopoietic cells (most likelymicroglia) (Hess et al., 2004; Unger et al., 1993) into the centralnervous system, where the missing enzyme is expressed by engrafted cellsfrom which point the enzyme diffuses into CNS tissues and participatesin clearing of storage materials. The level of enzyme provided to CNStissues is thus limited to that amount expressed and released fromdonor-derived cells engrafting in the brain. While such engraftment isof great benefit for MPS I, recipients nonetheless continue to exhibitbelow normal IQ and impaired neurocognitive capability (Ziegler andShapiro, 2007).

The phenomenon of metabolic cross correction also explains theeffectiveness of ERT for several lysosomal storage diseases (Brady,2006), most notably MPS I. However, due to the requirement forpenetration of the blood-brain barrier (BBB) by the enzyme missing inthe particular lysosomal storage disease (LSD) in order to effectivelyreach the CNS, effectiveness of enzyme therapy for neurologicmanifestations of lysosomal storage disease (LSD) has not been observed(Brady, 2006). Enzymes are almost always too large and generally toocharged to effectively cross the BBB. This has prompted investigationsinto invasive intrathecal enzyme administration (Dickson et al., 2007),for which effectiveness has been demonstrated in a canine model of MPS I(Kakkis et al., 2004) and for which human clinical trials are beginningfor MPS I (Pastores, 2008; Munoz-Rojas et al., 2008). Key disadvantagesof enzyme therapy include its great expense $200,000 per year) and therequirement for repeated infusions of recombinant protein. Currentclinical trials of intrathecal IDUA administration are designed toinject the enzyme only once every three months, so the effectiveness ofthis dosing regimen remains uncertain.

SUMMARY OF THE INVENTION

Methods of preventing, inhibiting, and/or treating one or more symptomsassociated with a disease of the central nervous system (CNS) in amammal in need thereof are described. The methods involve delivering tothe CNS of a mammal in need of treatment a composition comprising aneffective amount of a recombinant adeno-associated virus (rAAV) vectorcomprising an open reading frame encoding a gene product, e.g., atherapeutic gene product. Target gene products that may be encoded by anrAAV vector include, but are not limited to, alpha-L-iduronidase,iduronate-2-sulfatase, heparan sulfate sulfatase,N-acetyl-alpha-D-glucosaminidase, beta-hexosaminidase,alpha-galactosidase, betagalactosidase, beta-glucuronidase orglucocerebrosidase. Diseases that may be prevented, inhibited or treatedusing the methods disclosed herein include, but are not limited to,mucopolysaccharidosis type I disorder, a mucopolysaccharidosis type IIdisorder, or a mucopolysaccharidosis type VII disorder. The AAV vectorcan be administered in a variety of ways to ensure that it is deliveredto the CNS/brain, and that the transgene is successfully transduced inthe subject's CNS/brain. Routes of delivery to the CNS/brain include,but are not limited to intrathecal administration, intracranialadministration, e.g., intracerebroventricular administration, or lateralcerebro ventricular administration), intranasal administration,endovascular administration, and intraparenchymal administration.

In one embodiment, the methods involve delivering to the CNS of an adultmammal in need of treatment a composition comprising an effective amountof a rAAV-9 vector comprising an open reading frame encoding a gene. Inone embodiment, the methods involve delivering to the CNS of an adultmammal in need of treatment a composition comprising an effective amountof a rAAV-9 vector comprising an open reading frame encoding IDUA. Thesemethods are based, in part, on the discovery that an AAV-9 vector canefficiently transduce the therapeutic transgene in the brain/CNS ofadult subjects, restoring enzyme levels to wild type levels, (see FIG.15, infra). The results achieved using AAV-9 are surprising in view ofprevious work which demonstrated that intravascular delivery of AAV-9 inadult mice does not achieve widespread direct neuronal targeting (seeFoust et al., 2009), as well as additional data showing that directinjection of AAV8-IDUA into the CNS of adult IDUA-deficient miceresulted in poor transgene expression (FIG. 18). As proof of principle,the working examples described herein use a pre-clinical model for thetreatment of MPSI, an inherited metabolic disorder caused by deficiencyof the lysosomal enzyme alpha-L-iduronidase (IDUA). The working examplessurprisingly demonstrate that direct injection of AAV9-IDUA into the CNSof immunocompetent adult IDUA-deficient mice resulted in IDUA enzymeexpression and activity that is the same or higher than IDUA enzymeexpression and activity in wild-type adult mice (see FIG. 15, infra).

In an additional embodiment of the invention, the working examples alsodemonstrate that co-therapy to induce immunosuppression orimmunotolerization, or treatment of immunodeficient animals, can achieveeven higher levels of IDUA enzyme expression and activity. In anembodiment, patients with genotypes that promote an immune response thatneutralizes enzyme activity (see, e.g., Barbier et al., 2013) aretreated with an immunosuppressant in addition to the rAAV vectorcomprising an open reading frame encoding a gene product, such as IDUA.

Neonatal IDUA^(-/-) mice are not immunocompetent. However administrationof IDUA expressing AAV-8 to neonatal IDUA^(-/-) mice resulted in IDUAexpression (Wolf et al., 2011), thus tolerizing the animals to IDUA. Asdescribed herein, the applicability of AAV-mediated gene transfer toadult (immunocompetent) mice by direct infusion of AAV to the centralnervous system was shown using different routes of administration. Forexample, AAV-IDUA serotype 9 was administered by direct injection intothe lateral ventricles of adult IDUA-deficient mice that were eitherimmunocompetent, immunodeficient (NODSCID/IDUA-/-), immunosuppressedwith cyclophosphamide (CP), or immunotolerized by weekly injection ofhuman iduronidase protein (Aldurazyme) starting at birth. CPimmunosuppressed animals were also administered AAV9-IDUA by intranasalinfusion, by intrathecal injection, and by endovascular infusion withand without mannitol to disrupt the blood-brain barrier. Animals weresacrificed at 8 weeks after vector administration, and brains wereharvested and microdissected for evaluation of IDUA enzymatic activity,tissue glycosaminoglycans, and IDUA vector sequences in comparison withnormal and affected control mice. Results from these studies show thatnumerous routes for AAV vector administration directly to the CNS may beemployed, e.g., so as to achieve higher levels of protein deliveryand/or enzyme activity in the CNS. In addition, although the brain is animmunopriviledged site, administration of an immunosuppressant orimmunotolerization may increase the activity found in the brain afterAAV administration. Higher levels of expression per administrationand/or less invasive routes of administration are clinically morepalatable to patients.

Thus, the invention includes the use of recombinant AAV (rAAV) vectorsthat encode a gene product with therapeutic effects when expressed inthe CNS of a mammal. In one embodiment, the mammal is an immunocompetentmammal with a disease or disorder of the CNS (a neurologic disease). An“immunocompetent” mammal as used herein is a mammal of an age where bothcellular and humoral immune responses are elicited after exposure to anantigenic stimulus, by upregulation of Th1 functions or IFN-γ productionin response to polyclonal stimuli, in contrast to a neonate which hasinnate immunity and immunity derived from the mother, e.g., duringgestation or via lactation. An adult mammal that does not have animmunodeficiency disease is an example of an immunocompetent mammal. Forexample, an immunocompetent human is typically at least 1, 2, 3, 4, 5 or6 months of age, and includes adult humans without an immunodeficiencydisease. In one embodiment, the AAV is administered intrathecally. Inone embodiment, the AAV is administered intracranially (e.g.,intracerebroventricularly). In one embodiment, the AAV is administeredintranasally, with or without a permeation enhancer. In one embodiment,the AAV is administered endovascularly, e.g., carotid arteryadministration, with or without a permeation enhancer. In oneembodiment, the mammal that is administered the AAV is immunodeficientor is subjected to immunotolerization or immune suppression, e.g., toinduce higher levels of therapeutic protein expression relative to acorresponding mammal that is administered the AAV but not subjected toimmunotolerization or immune suppression. In one embodiment, an immunesuppressive agent is administered to induce immune suppression. In oneembodiment, the mammal that is administered the AAV is not subjected toimmunotolerization or immune suppression (e.g., administration of theAAV alone provides for the therapeutic effect).

The invention provides a method to prevent, inhibit or treat one or moresymptoms associated with a disease or disorder of the central nervoussystem in a mammal in need thereof. The method includes intrathecally,e.g., to the lumbar region, or intracerebroventricularly, e.g., to thelateral ventricle, administering to the mammal a composition comprisingan effective amount of a rAAV vector comprising an open reading frameencoding a gene product, the expression of which in the central nervoussystem of the mammal prevents, inhibits or treats the one or moresymptoms. In one embodiment, the gene product is a lysosomal storageenzyme. In one embodiment, the mammal is an immunocompetent adult. Inone embodiment, the rAAV vector is an RAV-1, MV-2, ARV-3, AAV-4, AAV-5,AAV-6, AAV-7, AAV-8, MV rh10, or AAV-9 vector. In one embodiment, themammal is a human. In one embodiment, multiple doses are administered.In one embodiment, the composition is administered weekly, monthly ortwo or more months apart.

In one embodiment, the method includes intrathecally, e.g., to thelumbar region, administering to a mammal a composition comprising aneffective amount of a rAAV vector comprising an open reading frameencoding a gene product, the expression of which in the central nervoussystem of the mammal prevents, inhibits or treats the one or moresymptoms, and optionally administering a permeation enhancer. In oneembodiment, the permeation enhancer is administered before thecomposition. In one embodiment, the composition comprises a permeationenhancer. In one embodiment, the permeation enhancer is administeredafter the composition. In one embodiment, the gene product is alysosomal storage enzyme. In one embodiment, the mammal is animmunocompetent adult. In one embodiment, the rAAV vector is an AAV-1,AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV rh10, or AAV-9 vector. Inone embodiment, the mammal is a human. In one embodiment, multiple dosesare administered. In one embodiment, the composition is administeredweekly, monthly or two or more months apart. In one embodiment, themammal that is intrathecally administered the AAV is not subjected toimmunotolerization or immune suppression (e.g., administration of theAAV alone provides for the therapeutic effect). In one embodiment, themammal that is intrathecally administered the AAV is immunodeficient oris subjected to immunotolerization or immune suppression, e.g., toinduce higher levels of therapeutic protein expression relative to acorresponding mammal that is intrathecally administered the AAV but notsubjected to immunotolerization or immune suppression.

In one embodiment, the method includes intracerebroventricularly, e.g.,to the lateral ventricle, administering to an immunocompetent mammal acomposition comprising an effective amount of a rAAV vector comprisingan open reading frame encoding a gene product, the expression of whichin the central nervous system of the mammal prevents, inhibits or treatsthe one or more symptoms. In one embodiment, the gene product is alysosomal storage enzyme. In one embodiment, the rAAV vector is anAAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV rh10, or AAV-9vector. In one embodiment, the rAAV vector is not a rAAV-5 vector. Inone embodiment, the mammal is a human. In one embodiment, multiple dosesare administered. In one embodiment, the composition is administeredweekly, monthly or two or more months apart. In one embodiment, themammal that is intracerebroventricularly administered the AAV is notsubjected to immunotolerization or immune suppression (e.g.,administration of the AAV alone provides for the therapeutic effect). Inone embodiment, the mammal that is intracerebroventricularlyadministered the AAV is immunodeficient or is subjected toimmunotolerization or immune suppression, e.g., to induce higher levelsof therapeutic protein expression relative to a corresponding mammalthat is intracerebroventricularly administered the AAV but not subjectedto immunotolerization or immune suppression. In one embodiment, themammal is immunotolerized to the gene product before the compositioncomprising the AAV is administered.

Further provided is a method to prevent, inhibit or treat one or moresymptoms associated with a disease or disorder of the central nervoussystem in a mammal in need thereof. The method includes endovascularlyadministering to the mammal a composition comprising an effective amountof a rAAV vector comprising an open reading frame encoding a geneproduct, the expression of which in the central nervous system of themammal prevents, inhibits or treats the one or more symptoms, and aneffective amount of a permeation enhancer. In one embodiment, thecomposition comprises the permeation enhancer. In one embodiment, thepermeation enhancer comprises mannitol, sodium glycocholate, sodiumtaurocholate, sodium deoxycholate, sodium salicylate, sodium caprylate,sodium caprate, sodium lauryl sulfate, polyoxyethylene-9-laurel ether,or EDTA. In one embodiment, the gene product is a lysosomal storageenzyme. In one embodiment, the mammal is an immunocompetent adult. Inone embodiment, the rAAV vector is an AAV-1, AAV-2, AAV-3, AAV-4, AAV-5,AAV-6, AAV-7, AAV-8, AAV rh10, or AAV-9 vector. In one embodiment, therAAV vector is not a rAAV-5 vector. In one embodiment, the mammal is ahuman. In one embodiment, multiple doses are administered. In oneembodiment, the composition is administered weekly. In one embodiment,the composition is administered weekly, monthly or two or more monthsapart. In one embodiment, the mammal that is endovascularly administeredthe AAV is not subjected to immunotolerization or immune suppression(e.g., administration of the AAV provides for the therapeutic effect).In one embodiment, the mammal that is endovascularly administered theAAV is immunodeficient or is subjected to immunotolerization or immunesuppression, e.g., to induce higher levels of therapeutic proteinexpression relative to a corresponding mammal that is endovascularlyadministered the AAV but not subjected to immunotolerization or immunesuppression.

In one embodiment, the method includes intranasally administering to amammal a composition comprising an effective amount of a rAAV-9 vectorcomprising an open reading frame encoding a gene product, the expressionof which in the central nervous system of the mammal prevents, inhibitsor treats the one or more symptoms, and optionally administering apermeation enhancer. In one embodiment, intranasal delivery may beaccomplished as described in U.S. Pat. No. 8,609,088, the disclosure ofwhich is incorporated by reference herein. In one embodiment, thepermeation enhancer is administered before the composition. In oneembodiment, the composition comprises a permeation enhancer. In oneembodiment, the permeation enhancer is administered after thecomposition. In one embodiment, the gene product is a lysosomal storageenzyme. In one embodiment, the mammal is an immunocompetent adult. Inone embodiment, the mammal is a human. In one embodiment, multiple dosesare administered. In one embodiment, the composition is administeredweekly, monthly or two or more months apart. In one embodiment, themammal that is intranasally administered the AAV is not subjected toimmunotolerization or immune suppression. In one embodiment, the mammalthat is intranasally administered the AAV is subjected toimmunotolerization or immune suppression, e.g., to induce higher levelsof IDUA protein expression relative to a corresponding mammal that isintranasally administered the AAV but not subjected toimmunotolerization or immune suppression.

Also provided is a method to prevent, inhibit or treat one or moresymptoms associated with a disease of the central nervous system in amammal in need thereof. The method includes administering to the mammala composition comprising an effective amount of a rAAV vector comprisingan open reading frame encoding a gene product, the expression of whichin the central nervous system of the mammal prevents, inhibits or treatsthe one or more symptoms, and an immune suppressant. In one embodiment,the immune suppressant comprises cyclophosphamide. In one embodiment,the immune suppressant comprises a glucocorticoid, cytostatic agentsincluding an alkylating agent or an anti-metabolite such asmethotrexate, azathioprine, mercaptopurine or a cytotoxic antibiotic, anantibody, or an agent active on immunophilin. In one embodiment, theimmune suppressant comprises a nitrogen mustard, nitrosourea, a platinumcompound, methotrexate, azathioprine, mercaptopurine, fluorouracil,dactinomycin, an anthracyclin, mitomycin C, bleomycin, mithramycin,IL2-receptor-(CD25-) or CD3-directed antibodies, anti-IL-2 antibodies,cyclosporin, tacrolimus, sirolimus, IFN-β, IFN-γ, an opioid, or TNF-α(tumor necrosis factor-alpha) binding agents such as infliximab(Remicade), etanercept (Enbrel), or adalimumab (Humira). In oneembodiment, the rAAV and the immune suppressant are co-administered. Inone embodiment, the rAAV is administered before and optionally after theimmune suppressant. In one embodiment, the immune suppressant isadministered before the rAAV. In one embodiment, the rAAV and the immunesuppressant are intrathecally administered. In one embodiment, the rAAVand the immune suppressant are intracerebroventricularly administered.In one embodiment, the rAAV is intrathecally administered and the immunesuppressant is intravenously administered. In one embodiment, the geneproduct is a lysosomal storage enzyme. In one embodiment, the mammal isan adult. In one embodiment, the rAAV vector is an AAV-1, AAV-2, AAV-3,AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV rh10, or AAV-9 vector. In oneembodiment, the mammal is a human. In one embodiment, multiple doses areadministered. In one embodiment, the composition is administered weekly.In one embodiment, the composition is administered weekly, monthly ortwo or more months apart.

The invention also provides a method to prevent, inhibit or treat one ormore symptoms associated with a disease of the central nervous system ina mammal in need thereof. A mammal immunotolerized to a gene productthat is associated with the disease is administered a compositioncomprising an effective amount of a rAAV vector comprising an openreading frame encoding a gene product, the expression of which in thecentral nervous system of the mammal prevents, inhibits or treats theone or more symptoms. In one embodiment, the gene product is a lysosomalstorage enzyme. In one embodiment, the mammal is an adult. In oneembodiment, the rAAV vector is an AAV-1, AAV-2, AAV-3, AAV-4, AAV-5,AAV-6, AAV-7, AAV-8, AAV rh10, or AAV-9 vector. In one embodiment, themammal is a human. In one embodiment, multiple doses are administered.In one embodiment, the composition is administered weekly.

Gene products that may be encoded by rAAV vectors include, but are notlimited to, alpha-L-iduronidase, iduronate-2-sulfatase, heparan sulfatesulfatase, N-acetyl-alpha-D-glucosaminidase, beta-hexosaminidase,alpha-galactosidase, betagalactosidase, beta-glucuronidase,glucocerebrosidase, fibroblast growth factor-2 (FGF-2), brain derivedgrowth factor (BDGF), neurturin, glial derived growth factor (GDGF),tyrosine hydroxylase, dopamine decarboxylase, or alutamic aciddecarboxylase.

Diseases that have one or more neurologic symptoms that may beprevented, inhibited or treated using the methods disclosed hereininclude, but are not limited to, Adrenoleukodystrophy, Alzheimerdisease, Amyotrophic lateral sclerosis, Angelman syndrome, Ataxiatelangiectasia, Charcot-Marie-Tooth syndrome, Cockayne syndrome,Deafness, Duchenne muscular dystrophy, Epilepsy, Essential tremor,Fragile X syndrome, Friedreich's ataxia, Gaucher disease, Huntingtondisease, Lesch-Nyhan syndrome, Maple syrup urine disease, Menkessyndrome, Myotonic dystrophy, Narcolepsy, Neurofibromatosis,Niemann-Pick disease, Parkinson disease, Phenylketonuria, Prader-Willisyndrome, Refsum disease, Rett syndrome, Spinal muscular atrophy,Spinocerebellar ataxia, Tangier disease, Tay-Sachs disease, Tuberoussclerosis, Von Hippel-Lindau syndrome, Williams syndrome, Wilson'sdisease, or Zellweger syndrome. In one embodiment, the disease is alysosomal storage disease, e.g., a lack or deficiency in a lysosomalstorage enzyme. Lysosomal storage diseases include, but are not limitedto, mucopolysaccharidosis (MPS) diseases, for instance,mucopolysaccharidosis type I, e.g., Hurler syndrome and the variantsScheie syndrome and Hurier-Scheic syndrome (a deficiency inalpha-L-iduronidase); Hunter syndrome (a deficiency ofiduronate-2-sulfatase); mucopolysaccharidosis type III, e.g., Sanfilipposyndrome (A, B, C or D; a deficiency of heparan sulfate sulfatase,N-acetyl-alpha-D-glucosaminidase, acetyl CoA:alpha-glucosaminideN-acetyl transferase or N-acetylglucosamine-6-sulfate sulfatase);mucopolysaccharidosis type IV e.g., mucopolysaccharidosis type IV, e.g.,Morquio syndrome (a deficiency of galactosamine-6-sulfate sulfatase orbeta-galactosidase); mucopolysaccharidosis type VI, e.g., Maroteaux-Lamysyndrome (a deficiency of arylsulfatase B); mucopolysaccharidosis typeII; mucopolysaccharidosis type III (A, B, C or D; a deficiency ofheparan sulfate sulfatase, N-acetyl-alpha-D-glucosaminidase, acetylCoA:alpha-glucosaminide N-acetyl transferase orN-acetylglucosamine-6-sulfate sulfatase); mucopolysaccharidosis type IV(A or B; a deficiency of galactosamine-6-sulfatase andbeta-galatacosidase); mucopolysaccharidosis type VI (a deficiency ofarylsulfatase B); mucopolysaccharidosis type VII (a deficiency inbeta-glucuronidase); mucopolysaccharidosis type VIII (a deficiency ofglucosamine-6-sulfate sulfatase); mucopolysaccharidosis type IX (adeficiency of hyaluronidase); Tay-Sachs disease (a deficiency in alphasubunit of beta-hexosaminidase); Sandhoff disease (a deficiency in bothalpha and beta subunit of beta-hexosaminidase); GM1 gangliosidosis (typeI or type II); Fabry disease (a deficiency in alpha galactosidase);metachromatic leukodystrophy (a deficiency of aryl sulfatase A); Pompedisease (a deficiency of acid maltase); fucosidosis (a deficiency offucosidase); alpha-mannosidosis (a deficiency of alpha-mannosidase);beta-mannosidosis (a deficiency of beta-mannosidase), ceroidlipofuscinosis, and Gaucher disease (types I, II and III; a deficiencyin glucocerebrosidase), as well as disorders such as Hermansky-Pudlaksyndrome; Amaurotic idiocy; Tangier disease; aspartylglucosaminuria;congenital disorder of glycosylation, type Ia; Chediak-Higashi syndrome;macular dystrophy, corneal, 1; cystinosis, nephropathic; Fanconi-Bickelsyndrome; Farber lipogranulomatosis; fibromatosis; geleophysicdysplasia; glycogen storage disease I; glycogen storage disease Ib;glycogen storage disease Ic; glycogen storage disease III; glycogenstorage disease IV; glycogen storage disease V; glycogen storage diseaseVI; glycogen storage disease VII; glycogen storage disease 0;immunoosseous dysplasia, Schimke type; lipidosis; lipase b;mucolipidosis II; mucolipidosis II, including the variant form;mucolipidosis IV; neuraminidase deficiency with beta-galactosidasedeficiency; mucolipidosis I; Niemann-Pick disease (a deficiency ofsphingomyelinase); Niemann-Pick disease without sphingomyelinasedeficiency (a deficiency of a npcl gene encoding a cholesterolmetabolizing enzyme); Relsum disease; Sea-blue histiocyte disease;infantile sialic acid storage disorder; sialuria; multiple sulfatasedeficiency; triglyceride storage disease with impaired long-chain fattyacid oxidation; Winchester disease; Wolman disease (a deficiency ofcholesterol ester hydrolase); Deoxyribonuclease I-like 1 disorder;arylsulfatase E disorder; ATPase, H+ transporting, lysosomal, subunit 1disorder; glycogen storage disease IIb; Ras-associated protein rab9disorder; chondrodysplasia punctata 1, X-linked recessive disorder;glycogen storage disease VIII; lysosome-associated membrane protein 2disorder; Menkes syndrome; congenital disorder of glycosylation, typeIc; and sialuria. Replacement of less than 20%, e.g., less than 10% orabout 1% to 5% levels of lysosomal storage enzyme found in nondiseasedmammals, may prevent, inhibit or treat neurological symptoms such asneurological degeneration in mammals.

In one embodiment, the methods described herein involve delivering tothe CNS of an immunocompetent human in need of treatment a compositioncomprising an effective amount of a rAAV-9 vector comprising an openreading frame encoding a IDUA. Routes of administration to the CNS/braininclude, but are not limited to intrathecal administration, intracranialadministration, e.g., intracerebroventricular administration or lateralcerebro ventricular administration intranasal administration,endovascular administration, and intraparenchymal administration.

Other viral vectors may be employed in the methods of the invention,e.g., viral vectors such as retrovirus, lentivirus, adenovirus, semlikiforest virus or herpes simplex virus vectors.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Experimental design for iduronidase-deficient adult miceadministered IDUA-AAV either intracerebroventricularly (ICV) (rightlateral ventricle) or intrathecally (lumbar area). To prevent immuneresponse, animals were either immunosuppressed with cyclophosphamide(CP), immunotolerized at birth by intravenous administration of humaniduonidase protein (aldurazyme), or the injections (10 microliters,3×10¹¹ particles) were carried out in NOD-SCID immunodeficient mice thatwere also iduronidase deficient. Animals were sacrificed at theindicated time post-treatment, the brains were microdissected andextracts assayed for iduronidase activity.

FIGS. 2A-B. IDUA activity in immunodeficient, IDUA deficient animalsafter ICV injection.

FIGS. 3A-B. IDUA activity in immunocompetent animals administered AAVvector by ICV route and treated with cyclophosphamide.

FIGS. 4A-B. IDUA activity in immunocompetent animals administered AAVvector by IT route and treated with cyclophosphamide.

FIGS. 5A-B. IDUA activity in immunotolerized immunocompetent animalsadministered AAV vector ICV.

FIG. 6. Compilation of all mean levels of IDUA activity for side-by-sidecomparison.

FIGS. 7A-E. Data are grouped according the area of the brain. A)hippocampus, B) cerebellum, C) cortex, D) striatum, and E) rest ofbrain.

FIGS. 8A-D. Assay for GAG storage material in the different sections ofthe brain for all four of the test groups. A) Immunodeficient mice, ICVadministration. B) Immunocompetent mice, ICV administration and CPadministration. C) Immunocompetent mice, ICV administration andaldurazyme administration. D) Immunocompetent mice, IT administrationand CP administration.

FIG. 9. Schematic of experimental design. All mice were adult MPS1 (IDUAdeficient) mice injected with 10 microliters (3×10¹¹ particles) of AAV.

FIGS. 10A-B. Intracranial infusion of AAV9IDUA into immunodeficient MPSI mice. Adult animals were injected with 10¹¹ vector genomes andevaluated for iduronidase expression in the brain after 10 weeks. Enzymeactivity levels in the brain were significantly higher than in thebrains of wild type animals, and ranged from 30- to 300-fold higher thanwild type.

FIGS. 11A-B. Intracranial administration of AAV9IDUA in immunocompetent,IDUA deficient mice. Adult animals were injected with 10¹¹ vectorgenomes, and immunosuppressed by weekly injection of cyclophosphamide(CP). CP injections were terminated at 6 weeks post vector injection dueto poor health, and the animals were sacrificed at 8 weekspost-injection. Brains were microdissected and assayed for IDUA enzymeactivity.

FIGS. 12A-B. Intracranial infusion of AAV9IDUA into immunotolerized MPSI mice. MPS 1 mice were tolerized with either a single dose ofAldurazyme at birth or multiple doses administered weekly, starting atbirth. Mice were infused with vector at 4 months, and sacrificed at 11weeks after injection. Brains were microdissected and analyzed foriduronidase expression. Enzyme activities ranged from an average of 10-to 1000-fold higher than wild type levels.

FIGS. 13A-B. Intrathecal administration of AAV9IDUA in immunocompetent,IDUA deficient animals. Adult MPS I mice were injected with AAV9IDUAintrathecally, followed by a weekly immunosuppressive regimen ofcyclophosphamide. Animals were sacrificed at 11 weeks post-injection,and then brains and spinal cords were analyzed for IDUA enzyme activity.

FIGS. 14A-B. Intrathecal infusion of AAV9IDUA in immunotolerized MPS Imice. IDUA deficient animals were tolerized at birth with a single doseof Aldurazyme or multiple doses administered weekly starting at birth.At 4 months of age animals were infused intrathecally with AAV9IDUAvector, and at 10 weeks post-injection animals were sacrificed, brainsmicrodissected and assayed for iduronidase activity. There wasrestoration of enzyme activity in all parts of the brain, withactivities in the cerebellum ranging from 200- to 1500-fold higher thanwild type levels. Levels of enzyme activity in the olfactory bulb andcerebellum (to the right of the dashed line) correspond to the rightY-axis.

FIGS. 15A-B. Intrathecal infusion of AAV9IDUA in immunocompetent MPS Ianimals. Control MPS I animals were injected with AAV9IDUA vector, butwere not immunosuppressed nor immunotolerized. Animals were sacrificedat 11 weeks after vector injection, and then their brains were assayedfor iduronidase activity. Enzyme levels were restored to wild typelevels in all parts of the brain, but were significantly lower than inanimals that were either immunosuppressed or immunotolerized.

FIG. 16. Normalization of glycosaminoglycan (GAG) levels followingintracranial or intrathecal AAV9 infusion. AAV9IDUA was injectedintracranially or intrathecally into immunodeficient, immunosuppressedor immunotolerized MPS I mice as indicated, Animals were sacrificed 8-11weeks after injection, then the brains were microdissected and analyzedfor GAG levels. GAG storage was restored to wild type levels or close towild type in all groups analyzed, H=hippocampus, Cb=cerebellum,C=cortex, S=striatum, ThB=thalamus and brain stem.

FIGS. 17A-B. IDUA vector copies in brain. Microdissected brains wereanalyzed for IDUA vector sequences by QPCR. The copy numbers inintracranially (A) and intrathecally (B) injected mice correlate to thelevels of enzyme activity depicted in FIGS. 11 and 13.

FIG. 18. ICV infusion of AAV8-MCI into adult animals.

FIGS. 19A-B. Intranasal administration of AAV9/IDUA in immunocompetent,IDUA deficient animals. Adult MPS I mice were infused with AAV9/IDUAintranasally, followed by a weekly immunosuppressive regimen ofcyclophosphamide. Animals were sacrificed at 12 weeks post-injection andbrains were analyzed for IDUA enzyme activity.

FIG. 20. IDUA vector copies in brain. Microdissected brains wereanalyzed for IDUA vector sequences by QPCR. The copy numbers inintranasally injected mice correlate to the levels of enzyme in FIG. 19.

DETAILED DESCRIPTION OF THE INVENTION Definitions

As used herein, “individual” (as in the subject of the treatment) meansa mammal. Mammals include, for example, humans; non-human primates,e.g., apes and monkeys; and non-primates, e.g., dogs, cats, rats, mice,cattle, horses, sheep, and goats. Non-mammals include, for example, fishand birds.

The term “disease” or “disorder” are used interchangeably, and are usedto refer to diseases or conditions wherein lack of or reduced amounts ofa specific gene product, e.g., a lysosomal storage enzyme, plays a rolein the disease such that a therapeutically beneficial effect can beachieved by supplementing, e.g., to at least 1% of normal levels.

“Substantially” as the term is used herein means completely or almostcompletely; for example, a composition that is “substantially free” of acomponent either has none of the component or contains such a traceamount that any relevant functional properly of the composition isunaffected by the presence of the trace amount, or a compound is“substantially pure” is there are only negligible traces of impuritiespresent.

“Treating” or “treatment” within the meaning herein refers to analleviation of symptoms associated with a disorder or disease,“inhibiting” means inhibition of further progression or worsening of thesymptoms associated with the disorder or disease, and “preventing”refers to prevention of the symptoms associated with the disorder ordisease.

As used herein, an “effective amount” or a “therapeutically effectiveamount” of an agent of the invention e.g., a recombinant AAV encoding agene product, refers to an amount of the agent that alleviates, in wholeor in part, symptoms associated with the disorder or condition, or haltsor slows further progression or worsening of those symptoms, or preventsor provides prophylaxis for the disorder or condition, e.g., an amountthat is effective to prevent, inhibit or treat in the individual one ormore neurological symptoms.

In particular, a “therapeutically effective amount” refers to an amounteffective, at dosages and for periods of time necessary, to achieve thedesired therapeutic result. A therapeutically effective amount is alsoone in which any toxic or detrimental effects of compounds of theinvention are outweighed by the therapeutically beneficial effects.

A “vector” as used herein refers to a macromolecule or association ofmacromolecules that comprises or associates with a polynucleotide andwhich can be used to mediate delivery of the polynucleotide to a cell,either in vitro or in vivo. Illustrative vectors include, for example,plasmids, viral vectors, liposomes and other gene delivery vehicles. Thepolynucleotide to be delivered, sometimes referred to as a “targetpolynucleotide” or “transgene,” may comprise a coding sequence ofinterest in gene therapy (such as a gene encoding a protein oftherapeutic interest) and/or a selectable or detectable marker.

“AAV” is adeno-associated virus, and may be used to refer to the virusitsell or derivatives thereof. The term covers all subtypes, serotypesand pseudotypes, and both naturally occurring and recombinant forms,except where required otherwise. As used herein, the term “serotype”refers to an AAV which is identified by and distinguished from otherAAVs based on its binding properties, e.g., there are eleven serotypesof AAVs, AAV-1-AAV-11, including AAV-2, AAV-5, AAV-8. AAV-9 and AAVrh10, and the term encompasses pseudotypes with the same bindingproperties. Thus, for example, AAV-5 serotypes include AAV with thebinding properties of AAV-5, e.g., a pseudotyped AAV comprising AAV-5capsid and a rAAV genome which is not derived or obtained from AAV-5 orwhich genome is chimeric. The abbreviation “rAAV” refers to recombinantadeno-associated virus, also referred to as a recombinant AAV vector (or“rAAV vector”).

An “AAV virus” refers to a viral particle composed of at least one AAVcapsid protein and an encapsidated polynucleotide. If the particlecomprises a heterologous polynucleotide (i.e., a polynucleotide otherthan a wild-type AAV genome such as a transgene to be delivered to amammalian cell), it is typically referred to as “rAAV”. AAV “capsidprotein” incudes a capsid protein of a wild-type AAV, as well asmodified forms of an AAV capsid protein which are structurally and orfunctionally capable of packaging a rAAV genome and bind to at least onespecific cellular receptor which may be different than a receptoremployed by wild type AAV. A modified AAV capsid protein includes achimeric AAV capsid protein such as one having amino acid sequences fromtwo or more serotypes of AAV, e.g., a capsid protein formed from aportion of the capsid protein from AAV-5 fused or linked to a portion ofthe capsid protein from AAV-2, and a AAV capsid protein having a tag orother detectable non-AAV capsid peptide or protein fused or linked tothe AAV capsid protein, e.g., a portion of an antibody molecule whichbinds the transferrin receptor may be recombinantly fused to the AAV-2capsid protein.

A “pseudotyped” rAAV is an infectious virus having any combination of anAAV capsid protein and an AAV genome. Capsid proteins from any AAVserotype may be employed with a rAAV genome which is derived orobtainable from a wild-type AAV genome of a different serotype or whichis a chimeric genome, i.e., formed from AAV DNA from two or moredifferent serotypes, e.g., a chimeric genome having 2 inverted terminalrepeats (ITRs), each ITR from a different serotype or chimeric ITRs. Theuse of chimeric genomes such as those comprising ITRs from two AAVserotypes or chimeric ITRs can result in directional recombination whichmay further enhance the production of transcriptionally activeintermolecular concatamers. Thus, the 5′ and 3′ ITRs within a rAAVvector of the invention may be homologous, i.e., from the same serotype,heterologous, i.e., from different serotypes, or chimeric, i.e., an ITRwhich has ITR sequences from more than one AAV serotype.

rAAV Vectors

Adeno-associated viruses of any serotype are suitable to prepare rAAV,since the various serotypes are functionally and structurally related,even at the genetic level. All AAV serotypes apparently exhibit similarreplication properties mediated by homologous rep genes; and allgenerally bear three related capsid proteins such as those expressed inAAV2. The degree of relatedness is further suggested by heteroduplexanalysis which reveals extensive cross-hybridization between serotypesalong the length of the genome; and the presence of analogousself-annealing segments at the termini that correspond to ITRs. Thesimilar infectivity patterns also suggest that the replication functionsin each serotype are under similar regulatory control. Among the variousAAV serotypes, AAV2 is most commonly employed.

An AAV vector of the invention typically comprises a polynucleotide thatis heterologous to AAV. The poiynucleotide is typically of interestbecause of a capacity to provide a function to a target cell in thecontext of gene therapy, such as up- or down-regulation of theexpression of a certain phenotype. Such a heterologous polynucleotide or“transgene,” generally is of sufficient length to provide the desiredfunction or encoding sequence.

Where transcription of the heterologous polynucleotide is desired in theintended target cell, it can be operably linked to its own or to aheterologous promoter, depending for exampie on the desired level and/orspecificity of transcription within the target cell, as is known in theart. Various types of promoters and enhancers are suitable for use inthis context. Constitutive promoters provide an ongoing level of genetranscription, and may be preferred when it is desired that thetherapeutic or prophylactic polynucleotide be expressed on an ongoingbasis. Inducible promoters generally exhibit low activity in the absenceof the inducer, and are up-regulated in the presence of the inducer.They may be preferred when expression is desired only at certain timesor at certain locations, or when it is desirable to titrate the level ofexpression using an inducing agent. Promoters and enhancers may also betissue-specific: that is, they exhibit their activity only in certaincell types, presumably due to gene regulatory elements found uniquely inthose cells.

Illustrative examples of promoters are the SV40 late promoter fromsimian virus 40, the Baculovirus polyhedron enhancer/promoter element,Herpes Simplex Virus thymidine kinase (HSV tk), the immediate earlypromoter from cytomegalovirus (CMV) and various retroviral promotersincluding LTR elements. Inducible promoters include heavy metal ioninducible promoters (such as the mouse mammary tumor virus (rnMTV)promoter or various growth hormone promoters), and the promoters from T7phage which are active in the presence of T7 RNA polymerase. By way ofillustration, examples of tissue-specific promoters include varioussurfactin promoters (for expression in the lung), myosin promoters (forexpression in muscle), and albumin promoters (for expression in theliver). A large variety of other promoters are known and generallyavailable in the art, and the sequences of many such promoters areavailable in sequence databases such as the GenBank database.

Where translation is also desired in the intended target cell, theheterologous polynucleotide will preferably also comprise controlelements that facilitate translation (such as a ribosome binding site or“RBS” and a polyadenylation signal). Accordingly, the heterologouspolynucleotide generally comprises at least one coding regionoperatively linked to a suitable promoter, and may also comprise, forexample, an operatively linked enhancer, ribosome binding site andpoly-A signal. The heterologous polynucleotide may comprise one encodingregion, or more than one encoding regions under the control of the sameor different promoters. The entire unit, containing a combination ofcontrol elements and encoding region, is often referred to as anexpression cassette.

The heterologous polynucleotide is integrated by recombinant techniquesinto or in place of the AAV genomic coding region (i.e., in place of theAAV rep and cap genes), but is generally flanked on either side by AAVinverted terminal repeat (ITR) regions. This means that an ITR appearsboth upstream and downstream from the coding sequence, either in directjuxtaposition, e.g., (although not necessarily) without any interveningsequence of AAV origin in order to reduce the likelihood ofrecombination that might regenerate a replication-competent AAV genome.However, a single ITR may be sufficient to carry out the functionsnormally associated with configurations comprising two ITRs (see, forexample, WO 94/13788), and vector constructs with only one ITR can thusbe employed in conjunction with the packaging and production methods ofthe present invention.

The native promoters for rep are self-regulating, and can limit theamount of AAV particles produced. The rep gene can also be operablylinked to a heterologous promoter, whether rep is provided as part ofthe vector construct, or separately. Any heterologous promoter that isnot strongly down-regulated by rep gene expression is suitable; butinducible promoters may be preferred because constitutive expression ofthe rep gene can have a negative impact on the host cell. A largevariety of inducible promoters are known in the art; incuding, by way ofillustration, heavy metal ion inducible promoters (such asmetallothionein promoters); steroid hormone inducible promoters (such asthe MMTV promoter or growth hormone promoters); and promoters such asthose from T7 phage which are active in the presence of T7 RNApolymerase. One sub-class of inducible promoters are those that areinduced by the helper virus that is used to complement the replicationand packaging of the rAAV vector. A number of helper-virus-induciblepromoters have also been described, including the adenovirus early genepromoter which is inducible by adenovirus E1A protein; the adenovirusmajor late promoter; the herpesvirus promoter which is inducible byherpesvirus proteins such as VP16 or 1CP4; as well as vaccinia orpoxvirus inducible promoters.

Methods for identifying and testing helper-virus-inducible promotershave been described (see, e.g., WO 96/17947). Thus, methods are known inthe art to determine whether or not candidate promoters arehelper-virus-inducible, and whether or not they will be useful in thegeneration of high efficiency packaging cells. Briefly, one such methodinvolves replacing the p5 promoter of the AAV rep gene with the putativehelper-virus-inducible promoter (either known in the art or identifiedusing well-known techniques such as linkage to promoter-less “reporter”genes). The AAV rep-cap genes (with p5 replaced), e.g., linked to apositive selectable marker such as an antibiotic resistance gene, arethen stably integrated into a suitable host cell (such as the HeLa orA549 cells exemplified below). Cells that are able to grow relativelywell under selection conditions (e.g., in the presence of theantibiotic) are then tested for their ability to express the rep and capgenes upon addition of a helper virus. As an initial test for rep and/orcap expression, cells can be readily screened using immunofluorescenceto detect Rep and/or Cap proteins. Confirmation of packagingcapabilities and efficiencies can then be determined by functional testsfor replication and packaging of incoming rAAV vectors. Using thismethodology, a helper-virus-inducible promoter derived from the mousemetallothionein gene has been identified as a suitable replacement forthe p5 promoter, and used for producing high titers of rAAV particles(as described in WO 96/17947).

Removal of one or more AAV genes is in any case desirable, to reduce thelikelihood of generating replication-competent AAV (“RCA”). Accordingly,encoding or promoter sequences for rep, cap, or both, may be removed,since the functions provided by these genes can be provided in trans.

The resultant vector is referred to as being “defective” in thesefunctions. In order to replicate and package the vector, the missingfunctions are complemented with a packaging gene, or a pluralitythereof, which together encode the necessary functions for the variousmissing rep and/or cap gene products. The packaging genes or genecassettes are in one embodiment not flanked by AAV IIRs and in oneembodiment do not share any substantial homology with the rAAV genome.Thus, in order to minimize homologous recombination during replicationbetween the vector sequence and separately provided packaging genes, itis desirable to avoid overlap of the two polynuclectide sequences. Thelevel of homology and corresponding frequency of recombination increasewith increasing length of homologous sequences and with their level ofshared identity. The level of homology that will pose a concern in agiven system can be determined theoretically and confirmedexperimentally, as is known in the art. Typically, however,recombination can be substantially reduced or eliminated if theoverlapping sequence is less than about a 25 nucleotide sequence if itis at least 80% identical over its entire length, or less than about a50 nucleotide sequence if It is at least 70% identical over its entirelength. Of course, even lower levels of homology are preferable sincethey will further reduce the likelihood of recombination. It appearsthat, even without any overlapping homology, there is some residualfrequency of generating RCA. Even further reductions in the frequency ofgenerating RCA (e.g,, by nonhomologous recombination) can be obtained by“splitting” the replication and encapsidation functions of AAV, asdescribed by Allen et al., WO 98/27204).

The rAAV vector construct, and the complementary packaging geneconstructs can be implemented in this invention in a number of differentforms. Viral particles, plasmids, and stably transformed host cells canall be used to introduce such constructs into the packaging cell, eithertransiently or stably.

In certain embodiments of this invention, the AAV vector andcomplementary packaging gene(s), if any, are provided in the form ofbacterial plasmids, ARV particles, or any combination thereof. In otherembodiments, either the AAV vector sequence, the packaging gene(s), orboth, are provided in the form of genetically altered (preferablyinheritably altered) eukaryotic cells. The development of host cellsinheritably altered to express the AAV vector sequence, AAV packaginggenes, or both, provides an established source of the material that isexpressed at a reliable level.

A variety of different genetically altered cells can thus be used in thecontext of this invention. By way of illustration, a mammalian host cellmay be used with at least one intact copy of a stably integrated rAAVvector. An AAV packaging plasmid comprising at least an AAV rep geneoperably linked to a promoter can be used to supply replicationfunctions (as described in U.S. Pat. No. 5,658,776), Alternatively, astable mammalian cell line with an AAV rep gene operably linked to apromoter can be used to supply replication functions (see, e.g, Trempeet al., WO 95/13392); Burstein et al. (WO 98/23018); and Johnson et al.(U.S. Pat. No. 5,656,785). The AAV cap gene, providing the encapsidationproteins as described above, can be provided together with an AAV repgene or separately (see, e.g., the above-referenced applications andpatents as well as Allen et al. (WO 98/27204). Other combinations arepossible and included within the scope at this invention.

Pathways for Delivery

Despite the immense network of the cerebral vasculature, systemicdelivery of therapeutics to the central nervous system (CNS) is noteffective for greater than 98% of small molecules and for nearly 100% oflarge molecules (Partridge, 2005). The lack of effectiveness is due tothe presence of the blood-brain barrier (BBB), which prevents mostforeign substances, even many beneficial therapeutics, from entering thebrain from the circulating blood. While certain small molecule, peptide,and protein therapeutics given systemically reach the brain parenchymaby crossing the BBB (Banks, 2008), generally high systemic doses areneeded to achieve therapeutic levels, which can lead to adverse effectsin the body. Therapeutics can be introduced directly into the CNS byintracerebroventricular or intraparenchymal injections. Intranasaldelivery bypasses the BBB and targets therapeutics directly to the CNSutilizing pathways along olfactory and trigeminal nerves innervating thenasal passages (Frey II, 2002; Thorne et al., 2004; Dhanda et al.,2005).

Any route of rAAV administration may be employed so long as that routeand the amount administered are prophylactically or therapeuticallyuseful. In one example, routes of administration to the CNS includeintrathecal and intracranial. Intracranial administration may be to thecisterna magna or ventricle. The term “cisterna magna” is intended toinclude access to the space around and below the cerebellum via theopening between the skull and the top of the spine. The term “cerebralventricle” is intended to include the cavities in the brain that arecontinuous with the central canal of the spinal cord. Intracranialadministration is via injection or infusion and suitable dose ranges forintracranial administration are generally about 10³ to 10¹⁵ infectiousunits of viral vector per microliter delivered in 1 to 3000 microlitersof single injection volume. For instance, viral genomes or infectiousunits of vector per micro liter would generally contain about 10⁴, 10⁵,10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³, or 10¹⁴ viral genomes orinfectious units of viral vector delivered in about 10, 50, 100, 200,500, 1000, or 2000 microliters. It should be understood that theaforementioned dosage is merely an exemplary dosage and those of skillin the art will understand that this dosage may be varied. Effectivedoses may be extrapolated from dose-responsive curves derived from invitro or in vivo test systems.

The AAV delivered in the intrathecal methods of treatment of the presentinvention may be administered through any convenient route commonly usedfor intrathecal administration. For example, the intrathecaladministration may be via a slow infusion of the formulation for aboutan hour. Intrathecal administration is via injection or infusion andsuitable dose ranges for intrathecal administration are generaliy about10³ to 10¹⁵ infectious units of viral vector per microliter deliveredin, for example, 1 to 3000 microliters or 0.5 to 15 milliliters ofsingle injection volume. For instance, viral genomes or infectious unitsof vector per microliter would generally contain about 10⁴, 10⁵, 10⁶,10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³, or 10¹⁴ viral genomes orinfectious units of viral vector.

The therapy, if a lysosomal storage enzyme such as IDUA is expressed,results in the normalization of lysosomal storage granules in theneuronal and/or meningeal tissue of the subjects as discussed above. Itis contemplated that the deposition of storage granules is amelioratedfrom neuronal and glial tissue, thereby alleviating the developmentaldelay and regression seen in individuals suffering with lysosomalstorage disease. Other effects of the therapy may include thenormalization of lysosomal storage granules in the cerebral meningesnear the arachnoid granulation, the presence of which in lysosomalstorage disease result in high pressure hydrocephalus. The methods ofthe invention also may be used in treating spinal cord compression thatresults from the presence of lysosomnal storage granules in the cervicalmeninges near the cord at C1-C5 or elsewhere in the spinal cord. Themethods of the invention also are directed to the treatment of cyststhat are caused by the perivascular storage of lysosomal storagegranules around the vessels of the brain. In other embodiments, thetherapy also may advantageously result in normalization of liver volumeand urinary glycosaminoglycan excretion, reduction in spleen size andapnea/hypopnea events, increase in height and growth velocity inprepubertal subjects, increase in shoulder flexion and elbow and kneeextension, and reduction in tricuspid regurgitation or pulmonicregurgitation.

The intrathecal administration of the present invention may compriseintroducing the composition into the lumbar area. Any suchadministration may be via a bolus injection. Depending on the severityof the symptoms and the responsiveness of the subject to the therapy,the bolus injection may be administered once per week, once per month,once every 6 months or annually. In other embodiments, the intrathecaladministration is achieved by use of an infusion pump. Those of skill inthe art are aware of devices that may be used to effect intrathecaladministration of a composition. The composition may be intrathecallygiven, for example, by a single injection, or continuous infusion. Itshould be understood that the dosage treatment may be in the form of asingle dose administration or multiple doses.

As used herein, the term “intrathecal administration” is intended toinclude delivering a pharmaceutical composition directly into thecerebrospinal fluid of a subject, by techniques including lateralcerebroventricular injection through a burrhole or cistemal or lumbarpuncture or the like. The term “lumbar region” is intended to includethe area between the third and fourth lumbar (lower back) vertebrae and,more inclusively, the L2-S1 region of the spine.

Administration of a composition in accordance with the present inventionto any of the above mentioned sites can be achieved by direct injectionof the composition or by the use of infusion pumps. For injection, thecomposition can be formulated in liquid solutions, e.g., y inphysiologically compatible buffers such as Hank's solution, Ringer'ssolution or phosphate buffer. In addition, the enzyme may be formulatedin solid form and re-dissolved or suspended immediately prior to use.Lyophilized forms are also included. The injection can be, for example,in the form of a bolus injection or continuous infusion (e.g., usinginfusion pumps) of the enzyme.

In one embodiment of the invention, the rAAV is administered by lateralcerebro-ventricular injection into the brain of a subject. The injectioncan be made, for example, through a burr hole made in the subject'sskull. In another embodiment, the enzyme and/or other pharmaceuticalformulation is administered through a surgically inserted shunt into thecerebral ventricle of a subject. For example, the injection can be madeinto the lateral ventricles, which are larger, even though injectioninto the third and fourth smaller ventricles can also be made. In yetanother embodiment, the compositions used in the present invention areadministered by injection into the cistema magna or lumbar area of asubject.

While the exact mechanisms underlying intranasal drug delivery to theCNS are not entirely understood, an accumulating body of evidencedemonstrates that pathways involving nerves connecting the nasalpassages to the brain and spinal cord are important. In addition,pathways involving the vasculature, cerebrospinal fluid, and lymphaticsystem have been implicated in the transport of molecules from the nasalcavity to the CNS. It is likely that a combination of these pathways isresponsible, although one pathway may predominate, depending on theproperties of the therapeutic, the characteristics of the formulation,and the delivery device used.

Therapeutics can rapidly gain access to the CNS following intranasaladministration along olfactory nerve pathways leading from the nasalcavity directly to the CNS. Olfactory nerve pathways are a majorcomponent of intranasal delivery, evidenced by the fact that fluorescenttracers are associated with olfactory nerves as they traverse thecribriform plate (Jansson et al., 2002), drug concentrations in theolfactory bulbs are generally among the highest CNS concentrationsobserved (Thorne et al., 2004; Banks et al., 2004; Graff et al., 2005a);Nonaka et al., 2008; Ross et al., 2004; Ross et al., 2008; Thorne etal., 2008), and a strong, positive correlation exists betweenconcentrations in the olfactory epithelium and olfactory bulbs (Dhuriaet al., 2009a).

Olfactory pathways arise in the upper portion of the nasal passages, inthe olfactory region, where olfactory receptor neurons (ORNs) areinterspersed among supporting cells (sustentacular cells), microvillarcells, and basal cells. ORNs mediate the sense of smell by conveyingsensory information from the peripheral environment to the CNS (Clericoet al., 2003). Beneath the epithelium, the lamina propria contains mucussecreting Bowman's glands, axons, blood vessels, lymphatic vessels, andconnective tissue. The dendrites of ORNs extend into the mucous layer ofthe olfactory epithelium, while axons of these bipolar neurons extendcentrally through the lamina propria and through perforations in thecribriform plate of the ethmoid bone, which separates the nasal andcranial cavities. The axons of ORNs pass through the subarachnoid spacecontaining CSF and terminate on mitral cells in the olfactory bulbs.From there, neural projections extend to multiple brain regionsincluding the olfactory tract, anterior olfactory nucleus, piriformcortex, amygdala, and hypothalamus (Buck, 2000). In addition to ORNs,chemosensory neurons located at the anterior tip of the nasal cavity inthe Grueneberg ganglion lead into the olfactory bulbs (Fuss et al.,2005; Koos et al, 2005).

The unique characteristics of the ORNs contribute to a dynamic cellularenvironment critical for intranasal delivery to the CNS. Due to thedirect contact with toxins in the external environment, ORNs regenerateevery 3-4 weeks from basal cells residing in the olfactory epithelium(Mackay-Sim, 2003). Special Schwann cell-like cells called olfactoryensheathing cells (OECs) envelope the axons of ORNs and have animportant role in axonal regeneration, regrowth, and remyelination(Field et al., 2003; Li et al., 2005a; Li et al., 2005b). The OECscreate continuous, fluid-filled perineurial channels that,interestingly, remain open, despite the degeneration and regeneration ofORNs (Williams et al., 2004).

Given the unique environment of the olfactory epithelium, it is possiblefor intranasally administered therapeutics to reach the CNS viaextracellular or intracellular mechanisms of transport along olfactorynerves. Extracellular transport mechanisms involve the rapid movement ofmolecules between cells in the nasal epithelium, requiring only severalminutes to 30 minutes for a drug to reach the olfactory bulbs and otherareas of the CNS after intranasal administration (Frey II, 2002; Balinet al., 1986). Transport likely involves bulk flow mechanisms (Thorne etal., 2004; Thorne et al., 2001) within the channels created by the OECs.Drugs may also be propelled within these channels by the structuralchanges that occur during depolarization and axonal propagation of theaction potential in adjacent axons (Luzzati et al., 2004). Intracellulartransport mechanisms involve the uptake of molecules into ORNs bypassive diffusion, receptor-mediated endocytosis or adsorptiveendocytosis, followed by slower axonal transport, taking several hoursto days for a drug to appear in the olfactory bulbs and other brainareas (Baker et al., 1986; Broadwell et al., 1985; Kristensson et al.,1971). Intracellular transport in ORNs has been demonstrated for small,lipophilic molecules such as gold particles (de Lorenzo, 1970; Gopinathet al., 1978), aluminum salts (Peri et al., 1987), and for substanceswith receptors on ORNs such as WGA-HRP (Thorne et al., 1995; Baker etal., 1986; ltaya et al., 1986; Shipley, 1985). Intracellular mechanisms,while important for certain therapeutics, are not likely to be thepredominant mode of transport into the CNS. While some large molecules,such as galanin-like peptide (GALP), exhibit saturable transportpathways into the CNS (Nonaka et al., 2008), for other large moleculessuch as NGF and insulin-like growth factor-I (IGF-I), intranasaldelivery into the brain is nonsaturable and not receptor mediated (Thomeet al., 2004; Chen et al., 1998; Zhao et al., 2004),

An often overlooked but important pathway connecting the nasal passagesto the CNS involves the trigeminal nerve, which innervates therespiratory and olfactory epithelium of the nasal passages and entersthe CNS in the pons (Clerico et al., 2003; Graff et al., 2003).Interestingly, a small portion of the trigeminal nerve also terminatesin the olfactory bulbs (Schaefer et al., 2002). The cellular compositionof the respiratory region of the nasal passages is different from thatof the olfactory region, with ciliated epithelial cells distributedamong mucus secreting goblet cells. These cells contribute tomucociliary clearance mechanisms that remove mucus along with foreignsubstances from the nasal cavity to the nasopharynx. The trigeminalnerve conveys sensory information from the nasal cavity, the oralcavity, the eyelids, and the cornea, to the CNS via the ophthalmicdivision (V1), the maxillary division (V2), or the mandibular division(V3) of the trigeminal nerve (Clerico et al., 2003; Gray, 1978).Branches from the ophthalmic division of the trigeminal nerve provideinnervation to the dorsal nasal mucosa and the anterior portion of thenose, while branches of the maxillary division provide innervation tothe lateral walls of the nasal mucosa. The mandibular division of thetrigeminal nerve extends to the lower jaw and teeth, with no directneural inputs to the nasal cavity. The three branches of the trigeminalnerve come together at the trigeminal ganglion and extend centrally toenter the brain at the level of the pons, terminating in the spinaltrigeminal nuclei in the brainstem. A unique feature of the trigeminalnerve is that it enters the brain from the respiratory epithelium of thenasal passages at two sites: (1) through the anterior lacerated foramennear the pons and (2) through the cribriform plate near the olfactorybulbs, creating entry points into both caudal and rostral brain areasfollowing intranasal administration. It is also likely that other nervesthat innervate the face and head, such as the facial nerve, or othersensory structures in the nasal cavity, such as the Grueneberg ganglion,may provide entry points for intranasally applied therapeutics into theCNS.

Traditionally, the intranasal route of administration has been utilizedto deliver drugs to the systemic circulation via absorption into thecapillary blood vessels underlying the nasal mucosa. The nasal mucosa ishighly vascular, receiving its blood supply from branches of themaxillary, ophthalmic and facial arteries, which arise from the carotidartery (Clerico et al., 2003; Cauna, 1982). The olfactory mucosareceives blood from small branches of the ophthalmic artery, whereas therespiratory mucosa receives blood from a large caliber arterial branchof the maxillary artery (DeSesso, 1993). The relative density of bloodvessels is greater in the respiratory mucosa compared to the olfactorymucosa, making the former region an ideal site for absorption into theblood (DeSesso, 1993). The vasculature in the respiratory regioncontains a mix of continuous and fenestrated endothelia (Grevers et al,1987; Van Diest et al., 1979), allowing both small and large moleculesto enter the systemic circulation following nasal administration.

Delivery to the CNS following absorption into the systemic circulationand subsequent transport across the BBB is possible, especially forsmall, lipophilic drugs, which more easily enter the blood stream andcross the BBB compared to large, hydrophilic therapeutics such aspeptides and proteins.

Increasing evidence is emerging suggesting that mechanisms involvingchannels associated with blood vessels, or perivascular channels, areinvolved in intranasal drug delivery to the CNS. Perivascular spaces arebound by the outermost layer of blood vessels and the basement membraneof the surrounding tissue (Pollock et al., 1997). These perivascularspaces act as a lymphatic system for the brain, where neuron-derivedsubstances are cleared from brain interstitial fluid by enteringperivascular channels associated with cerebral blood vessels.Perivascular transport is due to bulk flow mechanisms, as opposed todiffusion alone (Cserr et al., 1981; Groothuis et al., 2007), andarterial pulsations are also a driving force for perivascular transport(Rennels et al., 1985; Rennels et al., 1985). Intranasally applied drugscan move into perivascular spaces in the nasal passages or afterreaching the brain and the widespread distribution observed within theCNS could be due to perivascular transport mechanisms (Thorne et al.,2004).

Pathways connecting the subarachnoid space containing CSF, perineurialspaces encompassing olfactory nerves, and the nasal lymphatics areimportant for CSF drainage and these same pathways provide access forintranasally applied therapeutics to the CSF and other areas of the CNS.Several studies document that tracers injected into the CSF in thecerebral ventricles or subarachnoid space drain to the underside of theolfactory bulbs into channels associated with olfactory nervestraversing the cribriform plate and reach the nasal lymphatic system andcervical lymph nodes (Bradbury et al., 1983; Flatterer et al., 2006;Johnston et al., 2004a); Kida et al., 1993; Walter et al., 2006a; Walteret al., 2006b). Drugs can access the CNS via these same pathways afterintranasal administration, moving from the nasal passages to the CSF tothe brain interstitial spaces and perivascular spaces for distributionthroughout the brain. These drainage pathways are significant in anumber of animal species (sheep, rabbits, and rats) accounting forapproximately 50% of CSF clearance (Bradbury et al., 1981; Boulton etal., 1999; Boulton et al., 1996; Cserr et al., 1992). Pathways betweenthe nasal passages and the CSF are still important and functional inhumans, evidenced by the fact that therapeutics are directly deliveredto the CSF following intranasal delivery, without entering the blood toan appreciable extent (Born et al, 2002). A number of intranasal studiesdemonstrate that drugs gain direct access to the CSF from the nasalcavity, followed by subsequent distribution to the brain and spinalcord. Many intranasally applied molecules rapidly enter the CSF, andthis transport is dependent on the lipophilicity, molecular weight, anddegree of ionization of the molecules (Dhanda et al., 2005; Born et al.,2002; Kumar et al., 1974; Sakane et al., 1995; Sakane et al., 1994; Wanget al., 2007). Assessing distribution into the CSF can provideinformation on the mechanism of intranasal delivery.

Optimal delivery to the CNS along neural pathways is associated withdelivery of the agent to the upper third of the nasal cavity (Hanson etal., 2008). Although a supine position may be employed another positionfor targeting the olfactory region is with the “praying to Mecca”position, with the head down-and-forward. A supine position with thehead angle at 70° or 90° may be suitable for efficient delivery to theCSF using a tube inserted into the nostrils to deliver the drug viaintranasal administration (van den Berg et al., (2002)).

For intranasal drug administration nose drops may be administered over aperiod of 10-20 minutes to alternating nostrils every 1-2 minutes toallow the solution to be absorbed into the nasal epithelium (Thorne etal., 2004; Capsoni et al., 2002; Ross et al., 2004; Ross et al., 2008;Dhuria et al., 2009a; Dhuria et al., 2009b; Francis et al., 2008;Martinez et al., 2008). This noninvasive method does not involveinserting the device into the nostril. Instead, drops are placed at theopening of the nostril, allowing the individual to sniff the drop intothe nasal cavity. Other administration methods in anesthetizedindividual involve sealing the esophagus and inserting a breathing tubeinto the trachea to prevent the nasal formulation from being swallowedand to eliminate issues related to respiratory distress (Chow et al.,1999; Chow et al., 2001; Fliedner et al., 2006; Dahlin et al., 2001).Flexible tubing can be inserted into the nostrils for localized deliveryof a small volume of the drug solution to the respiratory or olfactoryepithelia, depending on the length of the tubing (Chow et al., 1999; Vanden Berg et al., 2003; van den Berg et al., 2004a; Banks et al., 2004;van den Berg et al., 2002; Vyas et al., 2006a; Chariton et al., 2007a;Gao et al., 2007a).

Nasal delivery devices, such as sprays, nose droppers or needle-lesssyringes, may be employed to target the agent to different regions ofthe nasal cavity. OptiMist™ is a breath actuated device that targetsliquid or powder nasal formulations to the nasal cavity, including theolfactory region, without deposition in the lungs or esophagus(Djupesland et al., 2006). The ViaNase™ device can also be used totarget a nasal spray to the olfactory and respiratory epithelia of thenasal cavity. Nasal drops tend to deposit on the nasal floor and aresubjected to rapid mucociliary clearance, while nasal sprays aredistributed to the middle meatus of the nasal mucosa (Scheibe et al.,2008).

The immune suppressant or immunotolerizing agent may be administered byany route including parenterally. In one embodiment, the immunesuppressant or immunotolerizing agent may be administered bysubcutaneous, intramuscular, or intravenous injection, orally,intrathecally, intracranially, or intranasally, or by sustained release,e.g., using a subcutaneous implant. The immune suppressant orimmunotolerizing agent may be dissolved or dispersed in a liquid carriervehicle. For parenteral administration, the active material may besuitably admixed with an acceptable vehicle, e.g., of the vegetable oilvariety such as peanut oil, cottonseed oil and the like. Otherparenteral vehicles such as organic compositions using solketal,glycerol, formal, and aqueous parenteral formulations may also be used.For parenteral application by injection, compositions may comprise anaqueous solution of a water soluble pharmaceutically acceptable salt ofthe active acids according to the invention, desirably in aconcentration of 0.01-10%, and optionally also a stabilizing agentand/or buffer substances in aqueous solution. Dosage units of thesolution may advantageously be enclosed in ampules.

The composition, e.g., rAAV containing composition, immune suppressantcontaining composition or immunotolerizing composition, may be in theform of an injectable unit dose. Examples of carriers or diluents usablefor preparing such injectable doses include diluents such as water,ethyl alcohol, macrogol, propylene glycol, ethoxylated isostearylalcohol, polyoxyisostearyl alcohol and polyoxyethylene sorbitan fattyacid esters, pH adjusting agents or buffers such as sodium citrate,sodium acetate and sodium phosphate, stabilizers such as sodiumpyrosulfite, EDTA, thioalycolic acid and thiolactic acid, isotonicagents such as sodium chloride and glucose, local anesthetics such asprocaine hydrochloride and lidocaine hydrochloride. Furthermore usualsolubilizing agents and analgesics may be added. Injections can beprepared by adding such carriers to the enzyme or other active,following procedures well known to those of skill in the art. A thoroughdiscussion of pharmaceutically acceptable excipients is available inREMINGTON'S PHARMACEUTICAL SCIENCES (Mack Pub. Co., N.J. 1991). Thepharmaceutically acceptable formulations can easily be suspended inaqueous vehicles and introduced through conventional hypodermic needlesor using infusion pumps. Prior to introduction, the formulations can besterilized with, preferably, gamma radiation or electron beamsterilization.

When the immune suppressant or immunotolerizing agent is administered inthe form of a subcutaneous implant, the compound is suspended ordissolved in a slowly dispersed material known to those skilled in theart, or administered in a device which slowly releases the activematerial through the use of a constant driving force such as an osmoticpump. In such cases, administration over an extended period of time ispossible.

The dosage at which the immune suppressant or immunotolerizing agentcontaining composition is administered may vary within a wide range andwill depend on various factors such as the severity of the disease, theage of the patient, etc., and may have to be individually adjusted. Apossible range for the amount which may be administered per day is about0.1 mg to about 2000 mg or about 1 mg to about 2000 mg. The compositionscontaining the immune suppressant or immunotolerizing agent may suitablybe formulated so that they provide doses within these ranges, either assingle dosage units or as multiple dosage units. In addition tocontaining an immune suppressant, the subject formulations may containone or more rAAV encoding a therapeutic gene product.

Compositions described herein may be employed in combination withanother medicament. The compositions can appear in conventional forms,for example, aerosols, solutions, suspensions, or topical applications,or in lyophilized form.

Typical compositions include a rAAV, an immune suppressant, a permeationenhancer, or a combination thereof, and a pharmaceutically acceptableexcipient which can be a carrier or a diluent. For example, the activeagent(s) may be mixed with a carrier, or diluted by a carrier, orenclosed within a carrier. When the active agent is mixed with acarrier, or when the carrier serves as a diluent, it can be solid,semi-solid, or liquid material that acts as a vehicle, excipient, ormedium for the active agent. Some examples of suitable carriers arewater, salt solutions, alcohols, polyethylene glycols,polyhydroxyethoxylated castor oil, peanut oil, olive oil, gelatin,lactose, terra alba, sucrose, dextrin, magnesium carbonate, sugar,cyclodextrin, amylase, magnesium stearate, talc, gelatin, agar, pectin,acacia, stearic acid or lower alkyl ethers of cellulose, silicic acid,fatty acids, fatty acid amines, fatty acid monoglycerides anddiglycerides, pentaerythritol fatty acid esters, polyoxyethylene,hydroxymethylcellulose and polyvinylpyrrolidone. Similarly, the carrieror diluent can include any sustained release material known in the art,such as glyceryl monostearate or glyceryl distearate, alone or mixedwith a wax.

The formulations can be mixed with auxiliary agents which do notdeleteriously react with the active agent(s). Such additives can includewetting agents, emulsifying and suspending agents, salt for influencingosmotic pressure, buffers and/or coloring substances preserving agents,sweetening agents or flavoring agents. The compositions can also besterilized if desired.

If a liquid carrier is used, the preparation can be in the form of aliquid such as an aqueous liquid suspension or solution. Acceptablesolvents or vehicles include sterilized water, Ringer's solution, or anisotonic aqueous saline solution.

The agent(s) may be provided as a powder suitable for reconstitutionwith an appropriate solution as described above. Examples of theseinclude, but are not limited to, freeze dried, rotary dried or spraydried powders, amorphous powders, granules, precipitates, orparticulates. The composition can optionally contain stabilizers, pHmodifiers, surfactants, bioavailability modifiers and combinations ofthese. A unit dosage form can be in individual containers or inmulti-dose containers.

Compositions contemplated by the present invention may include, forexample, micelles or liposomes, or some other encapsulated form, or canbe administered in an extended release form to provide a prolongedstorage and/or delivery effect, e.g., using biodegradable polymers,e.g., polylactide-polyglycolide. Examples of other biodegradablepolymers include poly(orthoesters) and poly(anhydrides).

Polymeric nanoparticles, e.g., comprised of a hydrophobic core ofpolylactic acid (PLA) and a hydrophilic shell of methoxy-poly(ethyleneglycol) (MPEG), may have improved solubility and targeting to the CNS.Regional differences in targeting between the microemulsion andnanoparticle formulations may be due to differences in particle size.

Liposomes are very simple structures consisting of one or more lipidbilayers of amphiphilic lipids, i.e., phospholipids or cholesterol. Thelipophilic moiety of the bilayers is turned towards each other andcreates an inner hydrophobic environment in the membrane. Liposomes aresuitable drug carriers for some lipophilic drugs which can be associatedwith the non-polar parts of lipid bilayers if they fit in size andgeometry. The size of liposomes varies from 20 nm to few μm.

Mixed micelles are efficient detergent structures which are composed ofbile salts, phospholipids, tri, di- and monoglycerides, fatty acids,free cholesterol and fat soluble micronutrients. As long-chainphospholipids are known to form bilayers when dispersed in water, thepreferred phase of short chain analogues is the spherical micellarphase. A micellar solution is a thermodynamically stable system formedspontaneously in water and organic solvents. The interaction betweenmicelles and hydrophobic/lipophilic drugs leads to the formation ofmixed micelles (MM), often called swallen micelles, too. In the humanbody, they incorporate hydrophobic compounds with low aqueous solubilityand act as a reservoir for products of digestion, e.g. monoglycerides.

Lipid microparticles includes lipid nano- and microspheres. Microspheresare generally defined as small spherical particles made of any materialwhich are sized from about 0.2 to 100 μm. Smaller spheres below 200 nmare usually called nanospheres. Lipid microspheres are homogeneousoil/water microernulsions similar to commercially available fatemulsions, and are prepared by an intensive sonication procedure or highpressure emulsifying methods (grinding methods). The natural surfactantlecithin lowers the surface tension of the liquid, thus acting as anemulsifier to form a stable emulsion. The structure and composition oflipid nanospheres is similar to those of lipid microspheres, but with asmaller diameter.

Polymeric nanoparticles serve as carriers for a broad variety ofingredients. The active components may be either dissolved in thepolymetric matrix or entrapped or adsorbed onto the particle surface.Polymers suitable for the preparation of organic nanoparticles includecellulose derivatives and polyesters such as poly(lactic acid),poly(alycolic acid) and their copolymer. Due to their small size, theirlarge surface area/volume ratio and the possibility of functionalizationof the interlace, polymeric nanoparticles are ideal carrier and releasesystems. If the particle size is below 50 nm, they are no longerrecognized as particles by many biological and also synthetic barrierlayers, but act similar to molecularly disperse systems.

Thus, the composition of the invention can be formulated to providequick, sustained, controlled, or delayed release, or any combinationthereof, of the active agent after administration to the individual byemploying procedures well known in the art. In one embodiment, theenzyme is in an isotonic or hypotonic solution. In one embodiment, forenzymes that are not water soluble, a lipid based delivery vehicle maybe employed, e.g., a microemulsion such as that described in WO2008/049588, the disclosure of which is incorporated by referenceherein, or liposomes.

In one embodiment, the preparation can contain an agent, dissolved orsuspended in a liquid carrier, such as an aqueous carrier, for aerosolapplication. The carrier can contain additives such as solubilizingagents, e.g., propylene glycol, surfactants, absorption enhancers suchas lecithin (phosphatidylcholine) or cyclodextrin, or preservatives suchas parabens. For example, in addition to solubility, efficient deliveryto the CNS following intranasal administration may be dependent onmembrane permeability. For enzymes where paracellular transport ishindered due to size and polarity, improving membrane permeability mayenhance extracellular mechanisms of transport to the CNS along olfactoryand trigeminal nerves. One approach to modifying membrane permeabilitywithin the nasal epithelium is by using permeation enhancers, such assurfactants, e.g., lauroylcarnitine (LC), bile salts, lipids,cyclodextrins, polymers, or tight junction modifiers.

Generally, the active agents are dispensed in unit dosage form includingthe active ingredient together with a pharmaceutically acceptablecarrier per unit dosage. Usually, dosage forms suitable for nasaladministration include from about 125 μg to about 125 mg, e.g., fromabout 250 μg to about 50 mg, or from about 2.5 mg to about 25 mg, of thecompounds admixed with a pharmaceutically acceptable carrier or diluent.

Dosage forms can be administered daily, or more than once a day, such astwice or thrice daily. Alternatively dosage forms can be administeredless frequently than daily, such as every other day, or weekly, if foundto be advisable by a prescribing physician.

The invention will be described by the following non-limiting examples.

EXAMPLE I AAV Vector-Mediated Iduronidase Gene Delivery in a MurineModel of Mucopolysaccharidosis Type I: Comparing Different Routes ofDelivery to the CNS

Mucopolysaccharidosis type I (MPS I) is an inherited metabolic disordercaused by deficiency of the lysosomal enzyme alpha-L-iduronidase (IDUA).Systemic and abnormal accumulation of glycosaminoglycans is associatedwith growth delay, organomegaly, skeletal dysplasia, and cardiopulmonarydisease. Individuals with the most severe form of the disease (Hurlersyndrome) suffer from neurodegeneration, mental retardation, and earlydeath. The two current treatments for MPS I (hematopoietic stem celltransplantation and enzyme replacement therapy) cannot effectively treatall central nervous system (CNS) manifestations of the disease.

With respect to gene therapy, it was previously demonstrated thatintravascular delivery of AAV-9 in adult mice does not achievewidespread direct neuronal targeting (see Foust et al, 2009). Previouswork also showed that direct injection of AAV8-IDUA into the CNS ofadult IDUA-deficient mice resulted in a low frequency or a poor level oftransgene expression (see FIG. 18). The following examples, which use apre-clinical model for the treatment of MPSI, surprisingly demonstratethat direct injection of AAV9-IDUA into the CNS of immunocompetent adultIDUA-deficient mice resulted in IDUA enzyme expression and activity thatis the same or higher than IDUA enzyme expression and activity inwild-type adult mice (see FIG. 15, infra).

Methods

AAV9-IDUA preparation. The AAV-IDUA vector construct (MCI) has beenpreviously described (Wolf et al., 2011) (mCags promoter). AAV-IDUAplasmid DNA was packaged into AAV9 virions at the University of FloridaVector Core, yielding a titer of 3×10¹³ vector genomes per milliliter.

ICV infusions. Adult Idua-/- mice were anesthetized using a cocktail ofketamine and xylazine (100 mg ketamine+10 mg xylazine per kg) and placedon a stereotactic frame. Ten microliters of AAV9-IDUA were infused intothe right-side lateral ventricle (stereotactic coordinates AR 0.4, ML0.8, DV 2.4 mm from bregma) using a Hamilton syringe. The animals werereturned to their cages on heating pads for recovery.

Intrathecal infusions. Infusions into young adult mice were carried outby injection of 10 μL AAV vector containing solution between the L5 andL6 vertebrae 20 minutes after intravenous injection of 0.2 mL 25%mannitol.

Immunotolerization. Newborn IDUA deficient mice were injected throughthe facial temporal vein with 5μL containing 5.8 μg of recombinantiduronidase protein (Aldurazyme), and then the animals were returned totheir cage.

Cyclophosphamide immunosuppression. For immunosuppression, animals wereadministered cyclophosphamide once per week at a dose of 120 mg/kgstarting one day after infusion with AAV9-IDUA vector.

Animals. Animals were anesthetized with ketamine/xylazine (100 mgketamine+10 mg xylazine per kg) and transcardially perfused with 70 mL.PBS prior to sacrifice. Brains were harvested and microdissected on iceinto cerebellum, hippocampus, striatum, cortex, and brainstem/thalamus(“rest”), The samples were frozen on dry ice and then stored at −80° C.Samples were thawed and homogenized in 1 mL of PBS using a motorizedpestle and permeabilized with 0.1% Triton X-100, IDUA activity wasdetermined by fluorometric assay using 4MU-iduronide as the substrate.Activity is expressed in units (percent substrate converted to productper minute) per mg protein as determined by Bradford assay (BioRad).

Tissues. Tissue homogenates were clarified by centrifugation for 3minutes at 13,000 rpm using an Eppendorf tabletop centrifuge model 5415D(Eppendorf) and incubated overnight with proteinase K, DNase1, andRnase. GAG concentration was determined using the Blyscan SulfatedGlycosaminoglycan Assay (Accurate Chemical) according to themanufacturer's instructions.

Results

FIG. 1 shows the results for iduronidase-deficient mice that wereadministered AAV either intracerebroventricularly (ICV) or intrathecally(IT). To prevent immune response, animals were either immunosuppressedwith cyclophosphamide (CP), immunotolerized at birth by intravenousadministration of human iduonidase protein (aidurazyme), or theinjections were carried out in NOD-SCID immunodeficient mice that werealso iduronidase deficient. Animals were sacrificed at the indicatedtime post-treatment, the brains were microdissected and extracts assayedfor iduronidase activity.

FIG. 2 illustrates data for immunodeficient, IDUA deficient animalsinjected ICV with AAV-IDUA vector. Those animals exhibited high levelsof IDUA expression (10 to 100 times wild type) in all areas of thebrain, with the highest level observed in the brain stem and thalamus(“rest”).

Immunosuppressed animals administered AAV vector by ICV route had arelatively lower level of enzyme in the brain compared to theimmunodeficent animals (FIG. 3). Note that immunosuppression may havebeen compromised in these animals because CP was withdrawn 2 weeksbefore sacrifice due to poor health.

FIG. 4 shows data for immunosuppressed animals administered AAV vectorby the IT route. Immunotolerized animals administered AAV vector ICVexhibited widespread IDUA activity in all parts of the brain (FIG. 5),similar to that observed in the immunodeficient animals, indicating theeffectiveness of the immunotolerization procedure.

FIG. 6 is a compilation of all mean levels of IDUA activity forside-by-side comparison, and FIG. 7 is data grouped according the areaof the brain.

GAG storage material was assayed in the different sections of the brainfor all four of the test groups. For each group, the mean of eachportion of the brain is shown on the left, the values for each of theindividual animals is shown on the right (FIG. 8), IDUA deficientanimals (far left) contained high levels of GAG compared to wild typeanimals (magenta bar). GAG levels were at wild-type or lower than wildtype for all portions of the brain in all groups of AAV-treated animals.GAG levels were slightly although not significantly higher thanwild-type in cortex and brainstem of animals administered AAV9-IDUAintrathecally.

Conclusions

The results show high and widespread distribution of IDUA in the brainregardless of the route of delivery (ICV or IT) although IDUA expressionin striatum and hippocampus was lower in animals injected IT versus ICV.There appears to be an immune response since immune deficient mice havehigher levels of expression than immunocompetent mice. With regard toICV injection, when CP was withdrawn early, IDUA expression is lower. Inaddition, immunotolerization was effective in restoring high levels ofenzyme activity. Further, GAG levels were restored to normal in alltreated experimental groups of mice.

EXAMPLE II Methods

AAV9IDUA Preparation. AAV-IDUA plasmid was packaged into AAV9 virions ateither the University of Florida vector core, or the University ofPennsylvania vector core, yielding a titer of 1-3×10¹³ vector genomesper millilter.

ICV infusions. See Example I.

Intrathecal infusions. See Example I.

Immunotolerization. As in Example I except: for multiple tolerizations,newborn IDUA deficient mice were injected with the first dose ofAldurazyme in the facial temporal vein, followed by 6 weekly injectionsadministered intraperitoneally.

Cyclophosphamide immunosuppression. See Example I.

Animals. Animals were anesthetized with ketamine/xylazine (100 mgketamine+10 mg xylazine per kg) and transcardially perfused with 70 mLPBS prior to sacrifice. Brains were harvested and microdissected on iceinto cerebellum, hippocampus, striatum, cortex, and brainstem/thalamus(“rest”), The samples were frozen on dry ice and then stored at −80° C.

Tissue IDUA activity. Tissue samples were thawed and homogenized insaline in a tissue homogenizer. Tissue homogenates were clarified bycentrifugation at 15,000 rpm in a benchtop Eppendorf centrifuge at 4° C.for 15 minutes. Tissue lysates (supernatant) were collected and analyzedfor IDUA activity and GAG storage levels.

Tissue GAG levels. Tissue lysates were incubated overnight withProteinase K, RNase and DNase. GAG levels were analyzed using theBlyscan Sulfated Glycosaminoglycan Assay according to the manufacturer'sinstructions.

IDUA Vector copies. Tissue homogenates were used for DNA isolation andsubsequent QPCR, as described in Wolf et al. (2011).

Results

FIG. 9 illustrates the experimental design and groups. Animals wereadministered AAV9IDUA vector either by intracerebroventricular (ICV) orintrathecal (IT) infusion. Vector administration was carried out inNOD-SCID immunodeficient (ID) mice that were also IDUA deficient, or inIDUA deficient mice that were either immunosuppressed withcyclophosphamide (CP), or immunotolerized at birth by a single ormultiple injections of human iduronidase protein (Aldurazyme). The timesof treatment with vector and sacrifice are as indicated in FIG. 9. Allvector administrations were carried out in adult animals ranging in acefrom 3-4.5 months. Animals were injected with 10 μL of vector at a doseof 3×10¹¹ vector genomes per 10 microliters.

FIG. 10 shows IDUA enzyme activities in intracranially infused,immunodeficient, IDUA deficient mice. High levels of enzyme activitywere seen in all areas of the brain, ranging from 30- to 300-fold higherthan wild type levels. Highest enzyme expressions were seen in thalamusand brain stem, and in the hippocampus.

Animals that were injected intracranially and immunosuppressed withcyclophosphamide (CP) demonstrated significantly lower levels of enzymeactivity than other groups (FIG. 11). However, CP administration in thiscase had to be withdrawn 2 weeks prior to sacrifice due to poor healthof the animals.

IDUA enzyme levels in animals tolerized at birth with IDUA protein(Aldurazyme) and administered vector intracranially are depicted in FIG.12. All animals showed high enzyme levels in all parts of the brain thatranged from 10- to 1000-fold higher than wild type levels, similar tolevels achieved in immunodeficient animals, indicating the effectivenessof the immunotolerization procedure.

FIG. 13 depicts IDUA enzyme levels in mice that were injectedintrathecally and administered CP on a weekly basis. Elevated levels ofIDUA were observed in all parts of the brain, especially in thecerebellum and the spinal cord. Levels of enzyme were the lowest in thestriatum and hippocampus with activities at wild type levels.

IDUA deficient mice were tolerized with Aldurazyme as described, andinjected with vector intrathecally (FIG. 14). There was widespread IDUAenzyme activity in all parts of the brain, with highest levels ofactivity in the brain stem and thalamus, olfactory bulb, spinal cord andthe cerebellum. Similar to the data in FIG. 13, the lowest levels ofenzyme activity were seen in the striatum, cortex and hippocampus.

Control immunocompetent IDUA deficient animals were infused with vectorintrathecally, without immunosuppression or immunotolerization (FIG.15). The results indicate that although enzyme activities were at wildtype levels or slightly higher, they are significantly lower than whatwas observed in animals that underwent immunomodulation. The decreasesin enzyme levels were especially significant in the cerebellum,olfactory bulb and thalamus and brain stem, areas that expressed thehighest levels of enzyme in immunomodulated animals.

Animals were assayed for GAG storage material, as shown in FIG. 15. Allgroups demonstrated clearance of GAG storage, with GAG levels similar tothat observed in wild type animals. Animals that were immunosuppressedand injected with AAV-IDUA vector intrathecally had GAG levels in thecortex that were slightly higher than wild type, but still much lowerthan untreated IDUA deficient mice.

The presence of AAV9IDUA vector in animals that were immunotolerized andinjected with vector either intracranially or intrathecally wasevaluated by OPCR, as illustrated in FIG. 16. IDUA copies per cell werehigher in animals infused intracranially in comparison with animalsinfused intrathecally, which is consistent with the higher level ofenzyme activity seen in animals injected intracranially.

Conclusions

High, widespread, and therapeutic levels of IDUA were observed in allareas of the brain after intracerebroventricular and intrathecal routesof AAV9IDUA administration in adult mice. Enzyme activities wererestored to wild type levels or slightly higher in immunocompetent IDUAdeficient animals infused with AAV-IDUA intrathecally. Significantlyhigher levels of IDUA enzyme were observed for both routes of vectorinjection in animals immunotolerized starting at birth by administrationof IDUA protein.

EXAMPLE III

Adult immunocompetent IDUA deficient mice (12 weeks old) wereanesthetized with ketamine/xylazine, followed by intranasal infusion ofAAV9IDUA vector. Vector was administered by applying eight 3 μL dropswith a micropipette to the intranasal cavity, alternating betweennostrils, at 2 minute intervals between each application. A total of2.4-7×10¹¹ vector genomes was administered to each adult animal,depending on source of vector. Animals were immunosuppressed with 120mg/kg cyclophosphamide administered weekly, starting the day aftervector administration. Mice were sacrificed at 12 weeks post vectorinfusion, animals were assayed for IDUA enzyme expression and vectorcopies in the brain.

REFERENCES

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All publications, patents and patent applications are incorporatedherein by reference. While in the foregoing specification, thisinvention has been described in relation to certain preferredembodiments thereof, and many details have been set forth for purposesof illustration, it will be apparent to those skilled in the art thatthe invention is susceptible to additional embodiments and that certainof the details herein may be varied considerably without departing fromthe basic principles of the invention.

What is claimed is:
 1. A method to inhibit or treat one or more symptomsof Pompe Disease in a mammal in need thereof, comprising: administeringto a cisterna magna of the mammal in need thereof a compositioncomprising an amount of a recombinant adeno-associated virus (rAAV) 9 orrAAVrh10 vector comprising an open reading frame encoding acid maltaseeffective to inhibit or treat the one or more symptoms of Pompe Disease.2. The method of claim 1 wherein the mammal is an immunocompetent adult.3. The method of claim 1 wherein the mammal is a human.
 4. The method ofclaim 1 wherein neurodegeneration is inhibited or treated by theadministration.
 5. The method of claim 1 wherein prior to administrationof the composition the mammal is immunotolerized to acid maltase.
 6. Themethod of claim 1 wherein the amount administered reducesglycosaminoglycans (GAG).
 7. The method of claim 1 wherein rAAV9 vectoris administered.
 8. The method of claim 1 wherein rAAVrh10 vector isadministered.
 9. A method to inhibit or treat one or more symptoms ofPompe Disease in a mammal in need thereof, comprising: administering tothe mammal in need thereof an immune suppressant, and to a cisternamagna of the mammal a composition comprising an amount of a rAAV9 orrAAVrh10 vector comprising an open reading frame encoding acid maltaseeffective to inhibit or treat the one or more symptoms of Pompe Disease.10. The method of claim 9 wherein the immune suppressant comprisescyclophosphamide, a glucocorticoid, cytostatic agents including analkylating agent, an anti-metabolite, a cytotoxic antibiotic, anantibody, an agent active on immunophilin, a nitrogen mustard,nitrosourea, platinum compound, methotrexate, azathioprine,mercaptopurine, fluorouracil, dactinomycin, an anthracyclin e, mitomycinC, bleomycin, mithramycin, 1L-2 receptor-(CD25-) or CD3-directedantibodies, anti-IL-2 antibodies, ciclosporin, tacrolimus, sirolimus,IFN-beta, IFN-gamma, an opioid, or a TNF-alpha (tumor ecrosisfactor-alpha) binding agent.
 11. The method of claim 9 wherein the rAAVvector and the immune suppressant are co-administered or the immunesuppressant is administered after the rAAV vector.
 12. The method ofclaim 9 wherein the rAAV vector is a rAAV-9 vector.
 13. The method ofclaim 9 wherein multiple doses of the composition comprising the rAAV9or rAAVrh10 vector are administered.
 14. The method of claim 9 whereinthe rAAV vector is rAAVrh10 vector.
 15. The method of claim 9 whereinthe immune suppressant is administered before the rAAV9 or rAAVrh10vector.
 16. The method of claim 9 wherein the immune suppressant issystemically administered.
 17. The method of claim 9 wherein the mammalis a human.
 18. The method of claim 9 wherein the amount administeredreduces glycosaminoglycans (GAG).
 19. A method to inhibit or treat oneor more symptoms associated with a deficiency in acid maltase in amammal, comprising: providing a mammal with a deficiency in acid maltasethat is immunotolerized to acid maltase; and administering to a cisternamacro of the mammal a composition comprising an amount of a rAAV9 orrAAVrh10 vector comprising an open reading frame encoding acid maltaseeffective to inhibit or treat the one or more symptoms associated withthe deficiency in acid maltase.
 20. The method of claim 19 whereinmultiple doses of the composition comprising the rAAV9 or rAAVrh10vector are administered.
 21. The method of claim 19 wherein rAAV9 vectoris administered.
 22. The method of claim 19 wherein rAAVrh10 vector isadministered.
 23. The method of claim 19 wherein the mammal is a human.24. The method of claim 19 wherein the amount administered reducesglycosaminoglycans (GAG).